This invention relates to immunochemical indicator reagents and, more particularly, to such reagents capable of indicating the presence of specific antigenic or haptenic material by hemagglutination reaction therewith.
Agglutination testing and inhibition of agglutination testing are both well known techniques for indicating the presence of antibodies or antigens in a test specimen, such as a body fluid, secretion, exudate, transudate, tissue cells, a microorganism culture, or the like. Both of these techniques generally employ indicator reagents composed of a reactive moiety consisting of either antigens or antibodies, attached to suitable carrier particles for the purpose of providing the reagent with sufficient mass so that the product of the agglutination reaction may be readily detected with the unaided eye. In preparing such indicator reagents, the nature of the reactive moiety to be attached to the carrier particles will depend upon the type of material being detected, i.e., antibody or antigen, and the type of testing technique being employed, i.e., direct agglutination testing or inhibition of agglutination testing. For example, detection of specific antigenic material in a test specimen by direct agglutination testing would require an indicator reagent composed of carrier particles having attached thereto antibody globulin molecules having specific reactivity toward the specific antigenic material being detected, so that upon mixing the indicator reagent with the test specimen, the occurrence of an agglutination pattern would indicate the presence of the specific antigenic material being tested for, while the absence of an agglutination pattern would indicate the absence of that antigenic material.
Agglutination testing, being a relatively rapid, simple and economical procedure to perform, potentially has many advantages over other techniques currently commonly employed in clinical laboratories for the detection of specific antigenic material. Up to the present time, however, the technique of employing antibody-sensitized cells has not been widely employed for such purpose, due to the difficulties that have been encountered in preparing reliable indicator reagents having sufficient stability and sensitivity. The primary problem has been in obtaining an attachment of the antibody globulin molecules to the carrier particles which is sufficiently strong and stable so as to avoid leaching effect during storage or testing, without impairing the antigen-specific reactivity of the antibody globulin molecules. Thus, when attachment has been effected merely by physical adsorption of the antibody globulin molecules onto chemically inert carrier particles, such as quartz particles, bentonite particles, charcoal granules or latex particles, the antibody globulin molecules were found to leach from the surface of the carrier particles during storage in liquid suspension, freezing or lyophilization, as well as during the testing procedure itself, thereby leading to inaccurate test results. Moreover, previous attempts at using carrier particles having chemically reactive surface and effecting attachment of antibody globulin molecules thereto through chemical bonding have not produced bonds having a sufficiently high degree of strength and stability, and furthermore have generally resulted in an impairment of the antigen-specific reactivity of the antibody globulin molecules.
Of the chemically reactive carrier particles which have been proposed for this purpose, erythrocytes (red blood cells) are generally regarded as being the most suitable since they are readily available, their size is in the appropriate range to be detected in agglutination reactions by means of the unaided eye, their density is such that their rate of settling out of suspension produces agglutination reactions that can be read within a period of time that is practical (an hour or so), they are colored so their presence is easily detected, and their surface presents a large number and variety of chemical groups for possible reaction sites. In addition, the biconcave disc structure of erythrocytes and the extremely large number of plaques or raised areas on their surface further increase their effective surface area and provide the possibility of more chemically reactive sites being made available. Moreover, the one unsatisfactory feature of erythrocytes, their fragility, can be readily overcome by modifying the erythrocytes with some type of fixation, generally by treatment with an aldehyde fixing agent such as formaldehyde or glutaraldehyde.
The previous attempts at chemically bonding antibody globulin molecules to erythrocyte carrier particles have generally involved coupling reactions through the exposed aldehyde groups on the surface of aldehyde-fixed erythrocytes and exposed amino groups present in the antibody globulin molecules. Direct coupling of the two moieties in this manner has resulted in the antibody globulin molecules being bound too close to the surface of the erythrocyte carrier particles or falling into the crevices in the erythrocyte surface, leading to an impairment of the antigen-specific reactivity of the antibody globulin molecules due to steric hindrance effects. To avoid the steric hindrance effects, it has been found necessary to displace the antibody globulin molecules as far as possible from the surface of the erythrocyte carrier particles by means of a bifunctional coupling agent of substantial molecular size and having terminal reactivity with each of the two moieties being coupled. However, coupling agents meeting these requirements that have previously been tried for such purpose have not been found to be satisfactory due to their excessively high level of reactivity toward the antibody globulin molecules. For example, diazotized coupling agents, such as bis-diazotized benzidine and bis-diazotized dianisidine, which have been used with reasonable success in coupling antigens to erythrocyte carrier particles, have a destructive action toward antibody globulin molecules which impairs their antigen-specific reactivity, thereby resulting in indicator reagents having a relatively low degree of sensitivity. Moreover, the coupling action of these coupling agents to the erythrocyte surface, which is through the exposed aldehyde groups on the erythrocyte surface, results in chemical bonds whose degree of strength and stability is not exceptionally high and leaves substantial room for improvement.